2 Hensvold et al. Arthritis Research & Therapy (2015) 17:239 Page 2 of 10 Introduction Osteoimmunology is a conceptual and molecular understanding of how the immune system influences the bone metabolism in diseases such as rheumatoid arthritis (RA) [1, 2]. RA is a chronic inflammatory disease affecting the synovial membrane of the joints and bone [3, 4]. Approximately half of the patients, with symptom duration of less than 1 year, present with radiographic bone damage in small joints at diagnosis [5, 6]. Presence of systemic autoimmunity with rheumatoid factor (RF) and/or anti-citrullinated protein antibodies (ACPA) in RA is associated with an increased risk of bone damage [7 10]. Recently, a new cellular mechanism has been suggested by which ACPA specifically increase bone destruction in RA. According to this, ACPA binding to the surface of osteoclast precursors increases their number, possibly by stimulation of tumor necrosis factor alpha (TNF-α) production [11]. In addition to ACPA, markers of inflammation and of high disease activity (e.g., C-reactive protein (CRP) and disease activity score (DAS) 28) have also been shown to be associated with increased bone damage in patients with RA [8, 10]. Efficient treatment with diseasemodifying antirheumatic drugs (DMARD), including methotrexate (MTX), results in reduced disease activity and less bone destruction [12], while the effect on ACPA is still not completely elucidated [13, 14]. Receptor activator of nuclear factor kappa B ligand (RANKL) is in the concept of osteoimmunology; a key molecule in the regulation of bone metabolism and the linkage between immune and skeletal systems [15, 16]. RANKL is affected by proinflammatory cytokines such as TNF-α, interleukin (IL)-1 and IL-6 [4] and has been suggested to be a marker of bone damage in RA [17 20]. However, the linkage between immune system and the influence of ACPA immunity on RANKL in early RA is largely unexplored. RANKL is expressed in synovial tissue [18, 21, 22] and serum [6, 23, 24] but no studies on RANKL s relationship to ACPA status have been previously conducted in untreated RA. In this study, we aimed to determine to what extent RANKL levels associate with presence of ACPA, bone erosions and MTX treatment in a cohort of patients with early untreated RA. In summary, we can report that RANKL was elevated in ACPA-positive and in anti-cit-vimentin-positive patients and associated with bone erosions in patients with early untreated RA. Methods Patients The study was performed in a cohort of 183 patients with early untreated RA with symptom onset <1 year prior to diagnosis, recruited at the Rheumatology Clinic at Karolinska University Hospital, Stockholm (during years ) and part of the Epidemiological Investigation of Rheumatoid Arthritis (EIRA) study cohort [25]. Clinical data and data on rheumatoid factor (RF) positivity were obtained from the Swedish Rheumatology quality registers. All patients in this study started on MTX, with or without concomitant nonsteroidal antiinflammatory drugs (NSAID) and/or prednisolone, to a final dose of mg/week following the local guidelines. Regarding antiporotic treatment: 10 out of 181 patients (13 %, 2 with missing data) were on calcium and/or vitamin D supplements and 16 out of 181 (9 %, 2 with missing data) on hormone replacement therapy, while none was treated with either bisphosphonates or denosumab at inclusion. An additional number of 10 out of 181 patients (5 %) and 1 out of 181 patients (1 %) were prescribed calcium and/or vitamin D supplement, respectively, and/or bisphosphonates at inclusion. Serum samples and DAS28 based on the erythrocyte sedimentation rate (ESR) were obtained at baseline and at clinical follow-up, which occurred after a median of 14 weeks (interquartile range % (IQR) 13 15). Data on the presence of HLA-DRB1 shared epitope (SE) gene allele, protein tyrosine phosphatase gene allele (PTPN22 rs ) and body mass index (BMI) at inclusion were also available [26 28]. Review of the medical records provided information on the presence or absence of at least one bone erosion at baseline, based on standard radiographs of hands and feet according to clinical routine at our unit. Seven patients were excluded from posttreatment analyses due to delayed MTX treatment initiation (>7 weeks after baseline) or secession of treatment; these patients were, however, included in pretreatment (baseline) analyses. Additionally, 1116 patients from the EIRA study cohort with available study data on standard radiograph changes in hands (at least one change according to 1987 RA criteria [29]) and serum ACPA status [30], were selected as a validation cohort. Patients in this validation cohort had a median age of 54 years; 69 % were females, 62 % (n = 687) ACPA-positive and 36 % (n = 405) anticitrullinated vimentin (cit-vim)-positive. The Ethics Review Board at Karolinska Institutet approved the study and an informed consent was obtained from participants. Immunoassays ACPA were detected by anti-cyclic citrullinated peptide version 2 (anti-ccp2) test (CCPlus Immunoscan, Malmö, Sweden) using the manufacturer s protocol. ACPA levels above the highest standard sample (>3200 AU/ml) were reassessed after appropriate dilution. Antibodies (anti)-cit-enolase peptide 1 (CEP-1, amino acids 5 21), anti-cit-vim peptide (amino acids 60 75), and anti-cit-fibrinogen (fib) peptide (amino acids )

3 Hensvold et al. Arthritis Research & Therapy (2015) 17:239 Page 3 of 10 were measured in sera by previously established enzyme-linked immunosorbent assay (ELISA). Native forms of the same peptides were used as controls [31]. Serum concentrations of the total (bound and unbound) soluble RANKL were measured by ELISA (Biovendor, Brno, Czech Republic) in accordance with the manufacturer s instructions. Serum concentrations of CRP, IL-6, and tumor necrosis factor receptor type 1 (TNF-RI) were measured by multiplex sandwich immunoassay (Sector Imager 6000, Meso Scale Discovery, Gaithersburg, MD, USA) [32]. Paired samples (before and after treatment) were run at the same assay and handled thesameway. Synovial biopsies and immunohistochemistry Synovial biopsies obtained during arthroscopy were available from 15 patients with early untreated RA (including 7 of the 183 patients that donated blood). Patients had a median age of 56 years (range 33 78); 10 out of 15 were females; 7 out of 15 were ACPA-positive and 2 out of 15 had bone erosion at baseline. Synovial expression of RANKL was detected by immunohistochemistry staining, using a monoclonal anti-human RANKL detection antibody at a final concentration of 5 μg/ml (12A668, Abcam, Cambridge, UK) as previously described [22, 33]. The RANKL expression level was evaluated using computer-assisted image analysis and expressed as the percentage of the total tissue area that stained positive. Statistical analysis Pearson s chi-square test, Wilcoxon rank sum test and Wilcoxon signed rank test were used for independent and paired comparisons as appropriate. To investigate the relationship between RANKL and ACPA independent of RF, the analysis of RANKL was performed both in the overall group (n = 183) and also in the subset of RF-negative patients (n = 59). In order to investigate the association between RANKL concentration (logarithm-transformed concentration) and ACPA status, we also tested other possible predictors for RANKL: age, sex, smoking habits (history of ever or never smoking), BMI, DAS28-ESR, ESR, CRP, IL-6 serum levels, TNF-RI serum levels, health assessment questionnaire (HAQ) values, concurrent prednisolone usage, concurrent usage of antiporotic treatment, presence of HLA-DRB1 SE, presence of PTPN22 risk allele, one by one in univariate linear regression models. A multiple regression model for the association between RANKL and ACPA was then obtained by including the significant predictors from the univariate analyses. Statistical analyses were conducted using SAS 9.3 (SAS Institute Inc., Cary, NC, USA). Two tailed p values <0.05 were considered statistically significant. No adjustments for multiple testing were performed in the explorative part of the study, but Bonferroni correction was applied for comparisons in the validation cohort. Results Increased RANKL levels in ACPA-positive untreated early RA Of the 183 patients, 125 (68%) were ACPA-positive with a median concentration of 752 AU/ml (IQR ). Among these ACPA-positive patients 58% were also positive for anti-cit-enolase, 52% for anti-cit-vim, and 31% for anti-cit-fib (see Table 1). Serum concentration of RANKL (Fig. 1a) was significantly higher in ACPA-positive (689 pmol/l, IQR ) than in ACPA-negative patients (159 pmol/l, IQR , p <0.05). Similarly, median synovial expression of RANKL (Fig. 1b-d) was significantly higher in ACPApositive than in ACPA-negative patients. To further investigate the relationship between RANKL and ACPA independent of RF, we performed a separate analysis of RF-negative patients. Similar to the whole cohort, the median RANKL serum concentration remained significantly higher in RF-negative ACPApositive (222 pmol/l, IRQ , n = 14) as compared to RF-negative ACPA-negative patients (128 pmol/l, IRQ , n = 45, p <0.05) (Fig. 1e). Significantly higher RANKL concentrations were also observed for those who were anti-cit-enolase positive (272 pmol/l, IQR , n = 6) or anti-cit-vim positive (244 pmol/l, IQR , n = 7) compared with those who were anti-cit-enolase negative (146 pmol/l, IQR , n = 53, p <0.05) or anti-cit-vim negative (151pmol/l, IQR , n = 52, p <0.05), respectively. Using linear univariate regression models we identified significant association between serum RANKL and ACPA, age, DAS28-ESR and BMI, while all other tested variables (sex, smoking habits, ESR, CRP, IL-6 serum levels, TNF-RI serum levels, HAQ values, use of prednisolone or antiporotic treatment, presence of HLA- DRB1 SE and PTPN22 risk allele) were not significant predictors for RANKL. Only ACPA and BMI remained significant in the multivariate model. A mean of 232 pmol/l (95 % CI: ) RANKL in ACPA positive and 140 pmol/l (95 % CI: ) RANKL in ACPA negative was estimated after adjustments of age, DAS28- ESR and BMI (Table 2). Serum RANKL and ACPA associate with bone erosion in untreated early RA RANKL serum concentrations were significantly elevated (p <0.05) in patients with evidence of bone erosions at baseline (median 243 pmol/l, IQR , n = 9) compared with those without bone erosions (151 pmol/l, IQR , n = 50) (Fig. 2a). Baseline bone erosions were

7 Hensvold et al. Arthritis Research & Therapy (2015) 17:239 Page 7 of 10 A ACPA (AU/ml) Baseline * Follow-up B Anti-cit-enolase (AU/ml) Baseline * Follow-up C Anti-cit-vim (AU/ml) Baseline Baseline * Follow-up Follow-up D. D Anti-cit-fib (AU/ml) Baseline * Follow-up Fig. 3 Serum concentration of ACPA and ACPA specificities. Graphs show the results of ELISA measurements of the antibody concentrations for patients being positive at baseline or at 3 months for the corresponding antibody: ACPA n = 120 (a), anti-cit-enolase (amino acids 5 21) n = 71 (b), anti-citrullinated (cit)-vimentin (vim) (amino acids 60 75) n = 67 (c), anti-cit-fibrinogen (fib) (amino acids ) antibodies n = 42 (d). Horizontal lines represent median values, * p <0.05. Dotted lines delineate ELISA cutoff values for each antibody. ACPA anti-citrullinated protein antibodies, ELISA enzyme-linked immunosorbent assay presence of ACPA in RA. ACPA directly affects osteoclast precursors, but the relationship between ACPA and RANKL has not yet been investigated. In this study, we report that serum and synovial RANKL levels, used as surrogate makers of local and systemic bone destruction, are higher in ACPA-positive than in ACPA-negative newly diagnosed untreated RA patients. Both ACPA and RANKL levels decrease following treatment with methotrexate (MTX),. We show that RANKL serum levels are higher in ACPA-positive as compared to ACPA-negative patients with early untreated RA, in contrast to one previous study were they found difference in a degradation product of type I collagen (CTX-I) but not RANKL [34]. Detection of only free RANKL (in contrast to detection of both free and bound RANKL in our study) and/or inclusion of patients with treated established RA (in contrast to untreated early RA in our study) might explain this discrepancy. The results presented here are in line with a previous study showing higher levels of bone resorption markers (such as CTX-I, tartrate-resistant acid phosphatase 5b and cathespin K) in the serum of ACPApositive as compared to ACPA-negative newly diagnosed untreated RA patients [11]. Notably, the differences in RANKL serum levels depending on ACPA status in our study are still detectable when the analysis was performed in the subgroup of RF-negative patients, eliminating the bias of RF interference. We detected relatively low differences in RANKL levels between erosive and nonerosive disease (in median a 40 % lower level in erosive as compared to nonerosive disease) and following therapy (in median a 20 % decrease following 3 months of treatment). However, differences of similar magnitude have previously been shown to be of biological relevance for RA-associated bone destruction [6]. Equally important the decrease in RANKL levels after 3 months therapy with MYX was observed in a majority of the patients (>80%) and is in accordance with previous reports showing that MTX decrease RANKL expression in both synovial tissue and synovial-derived biopsies [22, 35, 36]. None of the measured inflammatory parameters (ESR, CRP, IL-6 or TNF-RI, data not shown) associated with ACPA and bone erosion in contrast to RANKL, which suggests an association between RANKL, ACPA and bone erosion that is at least partially uncoupled from inflammation. One previous investigation of serum pre-ra samples (n = 79) failed to detect differences in RANKL serum levels as compared to controls [37] despite more recent reports showing signs of bone destruction in ACPApositive individuals already before disease onset [38]. One possible explanation is that local bone metabolism and

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